Every year, significant portions of the world's commercially important agricultural crops, including foods, textiles, and various domestic plants are lost to pest infestation, resulting in losses in the millions of dollars. Various strategies have been used in attempting to control such pests.
One strategy is the use of broad spectrum pesticides, i.e., chemical pesticides with a broad range of activity. However, there are a number of disadvantages to using such chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, these chemical pesticides are frequently toxic to animals and humans, and targeted pests frequently develop resistance when repeatedly exposed to such substances.
Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. Biopesticides comprise a bacterium which produces a toxin, a substance toxic to the pest. Biopesticides are generally less harmful to non-target organisms and the environment as a whole than chemical pesticides.
The most widely used biopesticide is Bacillus thuringiensis (B.t.). B.t. is a widely distributed, rod shaped, aerobic and spore-forming microorganism. During its sporulation cycle, B.t. produces a protein(s) known as a crystal delta-endotoxin(s), which kills insect larvae. B.t., therefore, is very useful as an agricultural pesticide.
Some strains, e.g., Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai, have been found to be specific for Lepidoptera Bacillus thuringiensis subsp. israelensis has been found to be specific for Diptera (Goldberg, U.S. Pat. No. 4,166,112). Other strains, e.g., Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No. 4,766,203), have been found to be specific for Coleoptera. The isolation of another coleopteran toxic Bacillus thuringiensis strain was reported in 1986 (Hernnstadt et al. Bio/Technology vol. 4, 305-308, 1986, U.S. Pat. No. 4,764,372, 1988). This strain, designated "Bacillus thuringiensis subsp. san diego", M-7, has been deposited at the Northern Regional Research Laboratory, USA under accession number NRRL B-15939. However, the assignee of the '372 patent, Mycogen, Corp. has publicly acknowledged that Bacillus thuringiensis subsp. san diego is Bacillus thuringiensis subsp. tenebrionis. Furthermore, the '372 patent has been assigned to Novo Nordisk A/S. Additionally, there has been disclosed a B.t. strain which is toxic against Lepidoptera and Coleoptera (PCT Application No. WO 90/13651). The toxin disclosed in PCT Application No. WO 90/13651 has a molecular weight of 81 kd.
During its sporulation cycle, B.t. produces a protein(s) in crystal form known as a crystal delta-endotoxin(s), having a molecular weight ranging from 27-140 kd, which upon ingestion kills insect larvae. Toxic activity may reside in one or more of such delta-endotoxins in a given B.t. strain. Most delta-endotoxins are protoxins that are proteolytically converted into smaller toxic (truncated) polypeptides in the target insect midgut (Hofte and Whiteley, 1989, Microbiol. Rev. 53:242-255). The delta-endotoxins are encoded by cry (crystal protein) genes. The cry genes have been divided into six classes and several subclasses based on structural similarities and pesticidal specificity. The major classes are Lepidoptera-specific (cryI); Lepidoptera-and Diptera-specific (cryII); Coleoptera-specific (cryIII); Diptera-specific (cryIV) (Hofte and Whiteley, 1989, Microbiol. Rev. 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992, Molecular Microbiology 6:1211-1217); and Nematode-specific (referred to as cryV and cryVI by Feitelson et al., 1992, Bio/Technology 10:271-275) genes.
Delta-endotoxins have been produced by recombinant DNA methods. The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form.
B.t. delta-endotoxin is water insoluble except at alkaline pH, and is almost always plasmid encoded. Some strains of Bacillus thuringiensis have been shown to produce a heat-stable pesticidal adenine-nucleotide analog, known as .beta.-exotoxin or thuringiensin, which is pesticidal alone (Sebesta et al., in H. D. Burges (ed.), Microbial Control of Pests and Plant Diseases, Academic Press, New York p. 249-281, 1981). .beta.-exotoxin has been found in the supernatant of some Bacillus thuringiensis cultures. It has a molecular weight of 789 and is comprised of adenosine, glucose, and allaric acid (Luthy et al., in Kurstak (ed.), Microbial and Viral Pesticides, Marcel Dekker, New York, 1982, pp. 35-72). Its host range includes, but is not limited to, Musca domestica, Mamestra configurata Walker, Tetranychus urticae, Drosophila melanogaster, and Tetranychus cinnabarinus. The toxicity of .beta.-exotoxin is thought to be due to inhibition of DNA-directed RNA polymerase by competition with ATP. It has been shown that .beta.-exotoxin is encoded by a Cry plasmid in five Bacillus thuringiensis (B.t.) strains and that .beta.-exotoxin may be classified as type I or type II .beta.-exotoxin (Levinson et al., 1990, J. Bacteriol. 172:3172-3179). .beta.-exotoxin type I was found to be produced by B.t. subsp. thuringiensis serotype 1, B.t. subsp. tolworthi serotype 9, and B.t. subsp. darmstadiensis serotype 10. .beta.-exotoxin type II was found to be produced by B.t. subsp. morrisoni serotype 8ab and is active against Leptinotarsa decemlineata (Colorado potato beetle). Other water soluble substances that have been isolated from B.t. include alpha-exotoxin which is toxic against the larvae of Musca domestica (Luithy, 1980, FEMS Microbiol. Lett. 8:1-7); gamma-exotoxins, which are various proteolytic enzymes including lecithinases, chitinases, and proteases, the toxic effects of which are expressed only in combination with beta-exotoxin or delta-endotoxin (Forsberg et al., 1976, Bacillus thuringiensis: Its Effects on Environmental Quality, National Research Council of Canada, NRC Associate Committee on Scientific Criteria for Environmental Quality, Subcommittees on Pesticides and Related Compounds and Biological Phenomena); sigma exotoxin which has a structure similar to beta-exotoxin, and is also active against Leptinotarsa decemlineata (Argauer et al., 1991, J. Entomol. Sci. 26:206-213); and anhydrothuringiensin (Coll. Czechoslovak Chem. Comm. 40, 1775, 1975).
WO 94/09630 discloses a water soluble substance that enhances the activity of Bacillus thuringiensis var. kurstaki and Bacillus thuringiensis var. aizawai.
Stonard et al. (1994, In Natural and Engineered Pest Management Agents, Paul A. Mann, Robert M. Hollingworth, eds., ACS, Washington, D.C., pp. 25-36) discloses diabroticins having the structure ##STR1## 1 R, R.sub.1, R.sub.2 .dbd.H, R.sub.3 .dbd.OH Diabroticin A 2 R, R.sub.1, R.sub.2, R.sub.3 .dbd.H Diabroticin B
Diabroticins were isolated from B. subtilis and have activity against Diabrotica undecimpunctata, Leptinotarsa decemlineata, Anthomus grandis Boheman, mosquito larvae, Staphylococcus aureus, and Micrococcus lutea, but did not have activity against European corn borer, Escherichia coli, B. subtilis, and Pseudomonas aeruginosa. Activity against other pests were not disclosed in Stonard et al. Diabroticin A was also isolated from fermentation broths of B. cereus.
The art has strived to achieve increased mortality of B.t. formulations. Means have included searching for new strains with increased mortality, engineering present strains, and designing more effective formulations by combining B.t. spores and/or crystals with new pesticidal carriers or with chemical pesticides.
It is an object of the present invention to improve the insecticidal activity of known B.t. formulations.
It is also an object of the present invention to enhance the pesticidal activity of pesticides as well as find novel uses for known pesticidal products.
It is advantageous to isolate new strains of Bacillus thuringiensis to produce new substances so that there exists a wider spectrum of biopesticides for any given insect pest.